A Density Functional Theory Investigation of Cleavage Mechanism of Dimethylsulphoniopropionate by Lyase DddQ

Dimethylsulfide (DMS) is released from the dimethylmercaptopropionate (DMSP) lyase decomposition reaction, which is of great significance for the global sulfur cycle. Among the various DMSP lyases, DddQ is one of the most prevalent and effective enzymes, catalyzing the conversion of DMSP into DMS. Using density functional calculations, we performed a detailed investigation of the DddQ reaction employing a concerted β-elimination mechanism via Tyr131 or Tyr120 as a general base. The reaction involves several key processes, including the deprotonation of nucleophile base, hydrogen proton transfer on Cα, and Cβ-S bond cleavage. Notably, the Hα proton transfer step is a critical step in determining the reaction rate in both mechanisms.


Introduction
Single-celled phytoplankton and algae present in the ocean annually produce approximately 109 tons of dimethylmercaptopropionate (DMSP) [1] .DMSP plays a crucial role in serving as an intracellular penetrant, primarily functioning as an anti-stress and antioxidant precursor.DMSP in the ocean undergoes two primary metabolic pathways.The first metabolic pathway involves demethylation reactions, while the second pathway is catalyzed by lyases present in algae and various marine bacteria, resulting in the cleavage of DMSP and the production of dimethylsulfide (DMS) and acrylate (Figure 1) [2] .DddQ, one of the DMSP lyases, has a more widespread distribution in the ocean.In 2016, the crystal structure (PDB: 5JSP) of DddQ containing the substrate DMSP has been reported [3] , and the DMSP decomposition catalyzed by DddQ is generally assumed to be a β-elimination mechanism (Figure 1).However, there is disagreement about whether the nucleophile base is Tyr131 or Tyr120.In this paper, using the density functional theory (DFT) and the hybrid functional B3LYP, we calculated two competition mechanisms of DddQ using a chemical model constructed based on an Xray crystal structure (PDB: 5JSP).

Computational methods
The current investigation utilized the density functional theory (DFT) and D3-dispersion correction [4] , as implemented in the Gaussian 09 software package [5] .The 6-31+G(d,p) basis set was used for substrate DMSP except for the Zn was employed by the SDD basis set.The 6-31G(d,p) basis set was used for the residues in the first shell.On the basis of the optimized geometries, a larger basis set 6-311+G(2d,2p) is used to obtain more accurate energies (except the Zn atom) [6,7] .In order to maintain the spatial conformation of the substrate and residues, the truncated Cα and two H atoms were rigidly constrained to their respective crystallographic positions.The energies listed have been corrected for the dispersion, ZPE, and solvation effects, unless stated otherwise.This method has been systematically tested and successfully used in studying the reaction mechanism of numerous biological enzymes [8] .

Construction of different reaction models
A chemical model React′ (Figure 3) based on the crystal structure (PDB: 4LA2) [2] was constructed (Figure 2).The 2-(N-morpholino) ethanesulfonic acid (Mes) was manually replaced with substrate DMSP in this model.The chemical model contains the substrate DMSP, Zn, the certain amino acids in the first shell (His125, His163, Glu129), and the second shell of React′ includes 11 amino acids (Trp114, Trp178, Pro187, Ala112, Va176, Thr76, His123, Tyr90, Tyr92, Tyr110, and Tyr120), which primarily form a hydrophobic pocket that facilitates the release of generated products.It should be noted that the hydroxyl oxygen of Tyr120 is 4.7 Å away from the C α of DMSP, making it difficult to directly abstracts the substrate α-hydrogen (H α ).Tyr131 is mainly regarded as a general base involved in the reaction (I).React′ consists of 267 atoms with a total charge of +1.In addition, we constructed another model React (Figure 3) based on the crystal structure (PDB: 5JSP) [4] .In React, the distance between the functional OH in Tyr120 and the C α of DMSP is from 4.7 Å in React′ to 3.4 Å in React.Tyr120 is regarded as a general base in the catalytic reaction (II).
React′ and React exhibit differences in their coordination mode between metals and amino acids.In React′, the residues (His163, His125, Glu129) and substrate DMSP in the first shell adopt a tetrahedral coordination pattern with Zn.Tyr131 and both oxygen atoms of the Glu129 terminal carboxyl group are coordinated with Zn, resulting in a six-coordination pattern.The calculation indicates that React has an energy 2.5 kcal/mol lower than React'.Therefore, in the subsequent discussion of Mechanism I and Mechanism II, React with lower energy is considered the zero point of reference energy.

Mechanism I: Reaction of Tyr131 as a base
The optimal reaction temperature and pH for lyase DddQ were found to be 30°C and 8.0.The pK a values of Tyr131 and Tyr120 are 13.13 and 18.97 determined by calculation [9] .This suggests that both Tyr131 and Tyr120 exist in a protonated state and should be deprotonated.The optimized structure (after deprotonation) was defined as the intermediate Inta (Figure 4) and the Zn-O distance (between Tyr131 and Zn) was shortened from 2.56 Å (React′) to 2.00 Å (Inta), a reasonable distance for the coordination interaction.Additionally, the lengths of coordination bonds of Zn were slightly lengthened (Figure 4).This step of deprotonation has an endothermicity of 0.9 kcal/mol (Figure 5).
Starting from the Inta, a transition state TSa for the β-elimination reaction (Figure 4) has been optimized and calculated to be an imaginary frequency of 1280i cm -1 , with an energetic barrier of 13.9 kcal/mol (Figure 5).Proton transfer accompanies the breaking of the C β -S bond via TSa.In TSa, the key Zn-O distance is elongated to 2.25 Å from 2.00 Å in Inta, while the C β -S distance is also altered from 1.85 Å in Inta to 1.94 Å in TSa.This step leads to the production of dimethylsulfide (DMS) and acrylate, resulting in the formation of the complex Proda.

Mechanism II: Reaction of tyrosine Tyr120 as a general base
From React, the reaction proceeds through deprotonation of the Tyr120, leading to an intermediate Intb (Figure 6).In Intb, the key O-H α distance between the terminal hydroxyl O of Tyr120 and the H α in Cα is 2.30 Å, resulting in a reasonable distance for the proton transfer reaction.In terms of spatial configuration, Intb still keeps the six-coordination structure.
Next, a transition state TSb for the proton transfer and cleavage of the C β -S bond has been found with a frequency of 1346i cm -1 correspond.The reaction barrier is calculated to be an exothermicity of 4.1 kcal/mol (Figure 5).Upon breaking of the C β -S bond, yielding the final products of dimethylsulfide (DMS) and acrylates.During the reaction, this step is rate-limiting with a barrier of 15.1 kcal/mol.

Figure 1 .
Figure 1.Decomposition of DMSP into DMS and acrylate by DddQ.

Figure 2 .
Figure 2. The overall structure of DddQ (left) and a detailed view of its active site (right).

Figure 3 .
Figure 3.The 2D schematic picture of the chemical model React′(left), where the optimized structure of React′ and React is given (right).

Figure 4 .
Figure 4. Optimized structure of stagnation points for tyrosine Tyr131 as a general base (Mechanism I).

Figure 5 .
Figure 5. Potential energy profiles of mechanisms I and II.The energies have been corrected.

Figure 6 .
Figure 6.The optimal structure of stagnation points for tyrosine Tyr120 as a general base (Mechanism II).